Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo[a]pyrene-7R-trans-7,8-dihydrodiol
Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo[a]pyrene-7R-trans-7,8-dihydrodiol
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DOI：
10.1093/carcin/bgg088
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发表时间：
2003-07-01
期刊：
影响因子：
4.7
通讯作者：
Sutter, TR
中科院分区：
文献类型：
--
作者：
Mammen, JS;Pittman, GS;Sutter, TR
常见致癌物代谢的个体差异背后的遗传差异是癌症易感性的重要潜在来源。细胞色素 P450 1B1 (CYP1B1) 是激活普遍存在的环境致癌物苯并[a]芘 (B[a]P) 的核心酶，具有多种遗传变异。本研究调查了六种罕见突变和四种常见多态性对 B[a]P 代谢的影响。与先天性青光眼相关的五种错义突变（Gly61Glu、Gly365Trp、Asp374Asn、Pro437Leu 和 Arg469Tryp）显着降低了 CYP1B1 将 (-)苯并[a]芘-7R-反式-7,8-二氢二醇（B[a]P-7,8-二醇）转化为(+/-)苯并[a]芘-r-7,t-8-二氢二醇-9,10-环氧化物。当使用酿酒酵母微粒体表达系统进行体外测定时，这五种突变导致酶的活性为正常活性的 3-12%。 10 bp 缺失突变未产生可检测到的蛋白质或活性。相比之下，含有四种常见单核苷酸多态性（Arg48Gly、Ala199Ser、Val432Leu、Asn453Ser）的所有可能组合的蛋白质对 B[a]P-7,8-二醇代谢具有适度影响。 Michaelis-Menten 分析表明，两个等位基因 Arg48、Ala119、Val432、Ser453 (RAVS) 和 Arg48、Ala119、Leu432、Ser453 (RALS) 的 K-M 值比 Arg48、Ala119、Val432、Ser453 (RAVN) 低 2 倍：1.4 +/- 0.3 和 1.3分别为 +/- 0.4 muM 与 2.8 +/- 0.8 muM 相比 (P < 0.05)。然而，这些差异无法通过直接测量低底物浓度下的速率来证实。其他两个动力学参数 k(cat) 或 k(cat)/K-M 均没有显着差异。三个人群的等位基因频率分析显示，Ser453 变异在亚洲血统 (< 1%) 和非洲血统 (< 4%) 的个体中很少见，而在欧洲血统的个体 (16%) 中更常见。含有 Ser453 变体的单倍型并不常见；在我们的小群体中仅检测到 RALS。 RALS 等位基因在亚洲人中的比例为 0.5%，在欧洲人中的比例为 15%。我们的研究表明，CYP1B1 中罕见的、与疾病相关的突变显着降低了 B[a]P-7,8-二醇的酶代谢；然而，由于四种常见的单氨基酸多态性，我们的结果并未发现这种代谢有任何重大差异。
Genetic differences that underlie inter-individual variation in the metabolism of common carcinogens are important potential sources of cancer susceptibility. Cytochrome P450 1B1 (CYP1B1), a central enzyme in the activation of the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P), has several genetic variants. This study investigated six rare mutations and four common polymorphisms for their effects on B[a]P metabolism. Five missense mutations associated with congenital glaucoma (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu and Arg469Tryp) dramatically decreased the capacity of CYP1B1 to convert (-)benzo[a]pyrene-7R-trans-7,8-dihyrodiol (B[a]P-7,8-diol) to (+/-)benzo[a]pyrene-r-7,t-8-dihydrodiol-9,10-epoxides. These five mutations resulted in enzymes with 3-12% of normal activity when assayed in vitro using an Saccharomyces cerevisiae microsomal expression system. A 10 bp deletion mutation produced no detectable protein or activity. In contrast, proteins containing all possible combinations of four common single nucleotide polymorphisms (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on B[a]P-7,8-diol metabolism. Michaelis-Menten analysis suggested that two alleles, Arg48, Ala119, Val432, Ser453 (RAVS) and Arg48, Ala119, Leu432, Ser453 (RALS), have K-M values 2-fold lower than Arg48, Ala119, Val432, Ser453 (RAVN): 1.4 +/- 0.3 and 1.3 +/- 0.4 muM, respectively, compared with 2.8 +/- 0.8 muM (P < 0.05). However, these differences could not be confirmed with direct measurements of rate at low substrate concentration. There were no significant differences for either of two other kinetic parameters, k(cat) or k(cat)/K-M. Allele frequency analysis in three populations reveals the Ser453 variant is rare among those of Asian (< 1 %) and African ancestry (< 4 %), and more common in individuals of European ancestry (16%). Haplotypes containing the Ser453 variant were uncommon; only RALS was detectable in our small populations. The RALS allele occurred between 0.5% in Asians and 15 % in Europeans. Our study demonstrates that rare, disease-associated mutations in CYP1B1 significantly decrease the enzyme's metabolism of B[a]P-7,8-diol; however, our results do not identify any major differences in this metabolism due to four common single amino acid polymorphisms.